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Cover page of Rumen metagenome and metatranscriptome analyses of low methane yield sheep reveals a Sharpea-enriched microbiome characterised by lactic acid formation and utilisation

Rumen metagenome and metatranscriptome analyses of low methane yield sheep reveals a Sharpea-enriched microbiome characterised by lactic acid formation and utilisation

(2016)

Background

Enteric fermentation by farmed ruminant animals is a major source of methane and constitutes the second largest anthropogenic contributor to global warming. Reducing methane emissions from ruminants is needed to ensure sustainable animal production in the future. Methane yield varies naturally in sheep and is a heritable trait that can be used to select animals that yield less methane per unit of feed eaten. We previously demonstrated elevated expression of hydrogenotrophic methanogenesis pathway genes of methanogenic archaea in the rumens of high methane yield (HMY) sheep compared to their low methane yield (LMY) counterparts. Methane production in the rumen is strongly connected to microbial hydrogen production through fermentation processes. In this study, we investigate the contribution that rumen bacteria make to methane yield phenotypes in sheep.

Results

Using deep sequence metagenome and metatranscriptome datasets in combination with 16S rRNA gene amplicon sequencing from HMY and LMY sheep, we show enrichment of lactate-producing Sharpea spp. in LMY sheep bacterial communities. Increased gene and transcript abundances for sugar import and utilisation and production of lactate, propionate and butyrate were also observed in LMY animals. Sharpea azabuensis and Megasphaera spp. act as important drivers of lactate production and utilisation according to phylogenetic analysis and read mappings.

Conclusions

Our findings show that the rumen microbiome in LMY animals supports a rapid heterofermentative growth, leading to lactate production. We postulate that lactate is subsequently metabolised mainly to butyrate in LMY animals, producing 2 mol of hydrogen and 0.5 mol of methane per mol hexose, which represents 24 % less than the 0.66 mol of methane formed from the 2.66 mol of hydrogen produced if hexose fermentation was directly to acetate and butyrate. These findings are consistent with the theory that a smaller rumen size with a higher turnover rate, where rapid heterofermentative growth would be an advantage, results in lower hydrogen production and lower methane formation. Together with previous methanogen gene expression data, this builds a strong concept of how animal traits and microbial communities shape the methane phenotype in sheep.

Cover page of Development of High Throughput Processes for Constructing Illumina Libraries

Development of High Throughput Processes for Constructing Illumina Libraries

(2010)

As the demand of constructing Illumina libraries increases, we have started to modify the library construction protocol to adapt the use of multichannel pipette and 96-well plates. With the few simple modification steps, we have doubled the library production efficiency. These modifications include the shearing of DNA with Covaris E210, and the cleaning of enzymatic reactions and fragment size selection with SPRI beads and a magnetic plate holder. We have also designed a set of molecular barcodes to enable the sequencing of many libraries in parallel. The requirements of these barcodes include 4 bases, balanced GC, and at least 2 bases difference between barcodes. The barcode is attached to the adaptor so it does not require third sequencing primer and the barcoded library can be run on the same flowcell/run with other non-barcoded libraries. We have begun to assess the ability to assign reads and the potential bias towards certain barcodes after pooling different number of libraries. We have recently programmed the Biomek FX robot to carry out the library construction process. Although this process still require manual transfer of plates from robot to other work stations, the processing of 96 Illumina libraries takes approximately 6-8 hours. This semi-automated process represents a significant increase of library capacity comparing to the manual process. We will present the progress and the challenges of these scale-up processes.

Cover page of PyroTag: Pyrosequencing as a Powerful Tool to Study the Diversity of Microbial Community

PyroTag: Pyrosequencing as a Powerful Tool to Study the Diversity of Microbial Community

(2008)

At JGI, major efforts have been spent on using the 16S RNA sequencing to study the composition of bacterial community. Currently, 16S rRNA genes from a microbial community are amplified with conserved PCR primers and the mixed amplicon is cloned and sequenced using traditional Sanger sequencing method. Using Roche GS FLX amplicon sequencing method, individual amplicon can be sequenced directly on the platform, producing hundreds of thousand ~;250 bp tags of the full length 16S rRNA gene. We call this pipeline PyroTag. The benefit of the PyroTag technology includes higher resolution and less bias than traditional PCR and cloning based sequencing. The disadvantage is that the read length is short as we used FLX reagent kit for the study. The aim for this study is to systematically test the effect of amplicon length and 16S rRNA regions targeted on diversity coverage of complex microbial community. The goal is to develop a standard PyroTag pipeline to provide to broad range JGI users. Molecular barcode method has been used to distinguish different amplicon length and different targeted 16S regions. Strategy and detailed experiment design as well as preliminary results will be presented.

Cover page of Genome Sequence and Analysis of the Soil Cellulolytic Actinomycete Thermobifida fusca

Genome Sequence and Analysis of the Soil Cellulolytic Actinomycete Thermobifida fusca

(2007)

Thermobifida fusca is a moderately thermophilic soil bacterium that belongs to Actinobacteria. 3 It is a major degrader of plant cell walls and has been used as a model organism for the study of 4 secreted, thermostable cellulases. The complete genome sequence showed that T. fusca has a 5 single circular chromosome of 3642249 bp predicted to encode 3117 proteins and 65 RNA6 species with a coding density of 85 percent. Genome analysis revealed the existence of 29 putative 7 glycoside hydrolases in addition to the previously identified cellulases and xylanases. The 8 glycosyl hydrolases include enzymes predicted to exhibit mainly dextran/starch and xylan 9 degrading functions. T. fusca possesses two protein secretion systems: the sec general secretion 10 system and the twin-arginine translocation system. Several of the secreted cellulases have 11 sequence signatures indicating their secretion may be mediated by the twin-arginine12 translocation system. T. fusca has extensive transport systems for import of carbohydrates 13 coupled to transcriptional regulators controlling the expression of the transporters and14 glycosylhydrolases. In addition to providing an overview of the physiology of a soil 15 actinomycete, this study presents insights on the transcriptional regulation and secretion of16 cellulases which may facilitate the industrial exploitation of these systems.